Microorganism Mediated Liquid Fuels

ABSTRACT

Herein disclosed is a method for producing liquid hydrocarbon product, the method comprising disintegrating a hydrocarbon source; pretreating the disintegrated hydrocarbon source; solubilizing the disintegrated hydrocarbon source to form a slurry comprising a reactant molecule of the hydrocarbon source; admixing a biochemical liquor into the slurry, wherein the biochemical liquor comprises at least one conversion enzyme configured to facilitate bond selective photo-fragmentation of said reactant molecule of the hydrocarbon source, to form liquid hydrocarbons via enzyme assisted bond selective photo-fragmentation, wherein said conversion enzyme comprises reactive sites configured to restrict said reactant molecule such that photo-fragmentation favorably targets a preselected internal bond of said reactant molecule; separating the liquid hydrocarbons from the slurry, wherein contaminants remain in the slurry; and enriching the liquid hydrocarbons to form a liquid hydrocarbon product. Various aspects of such method/process are also discussed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/133,449, filed Dec. 18, 2013, which is a continuation-in-partapplication of U.S. patent application Ser. No. 12/620,245 filed Nov.17, 2009, which claims benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application No. 61/141,552 filed Dec. 30, 2008 andU.S. Provisional Patent Application No. 61/146,816 filed Jan. 23, 2009.The disclosures of these three applications are hereby incorporatedherein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

A portion of this work was supported by federal funding under contractnumber DOE/NETL award: DE-FE0001259.

BACKGROUND Field of the Invention

This invention relates to producing liquid fuels, specifically toin-situ or ex-situ coal to liquid conversion.

Background of the Invention

Coals can also be converted into liquid fuels like gasoline or diesel byseveral different processes. In a developing commercial process, thecoal is converted into a gas first, and then into a liquid, by using theFischer-Tropsch (FT) process. In the FT process, an indirect route, coalis first gasified to make syngas, a purified mixture of CO and H₂ gas.Next, FT catalysts are used to convert the syngas into lighthydrocarbons, like ethane, which are further processed into refinableliquid fuels. In addition to creating the fuels, syngas can also beconverted into methanol, which can be used as a fuel, or a fueladditive.

Alternatively, the coal may be converted directly to liquid fuels viahydrogenation processes. For example, the Bergius process, in which coalis liquefied by mixing it with hydrogen gas and heating the system.Several other direct liquefaction processes have been developed, such asthe Solvent Refined Coal (SRC) processes, which has spawned severalpilot plant facilities. Additionally, dried, pulverized coal mixed withroughly 1 wt % molybdenum catalysts may be hydrogenated by use of hightemperature and pressure synthesis gas produced. However, the syngasmust be produced in a separate gasifier.

However, these coal to liquid fuel processes involve the mining of thecoal from the ground. As is well documented, coal mining is a hazardousprocess, and many mines are forced into closure prior to the removal ofall useable products. Further, those mines that are operated safelyleave behind large columns of coal to support the ceiling and coalresidues in the mine walls. These sources of coal represent asignificant amount of energy that is left abandoned by miningoperations. Further, these untouched resources may be converted toliquid fuels for transportation purposes. As such, there is a need inthe industry for the removal of abandoned, low quality, or residual coalfrom mining operations, for use in the coal to liquid production.

SUMMARY

Herein disclosed is a method for producing liquid hydrocarbon product,the method comprising disintegrating a hydrocarbon source; pretreatingthe disintegrated hydrocarbon source; solubilizing the disintegratedhydrocarbon source to form a slurry comprising a reactant molecule ofthe hydrocarbon source; admixing a biochemical liquor into the slurry,wherein the biochemical liquor comprises at least one conversion enzymeconfigured to facilitate bond selective photo-fragmentation of saidreactant molecule of the hydrocarbon source, to form liquid hydrocarbonsvia enzyme assisted bond selective photo-fragmentation, wherein saidconversion enzyme comprises reactive sites configured to restrict saidreactant molecule such that photo-fragmentation favorably targets apreselected internal bond of said reactant molecule; separating theliquid hydrocarbons from the slurry, wherein contaminants remain in theslurry; and enriching the liquid hydrocarbons to form a liquidhydrocarbon product.

In an embodiment, disintegrating the hydrocarbon source comprisescomminution of the hydrocarbon source. In an embodiment, comminutioncomprises grinding. In an embodiment, comminution compriseshigh-pressure steam treatment. In an embodiment, pretreating thedisintegrated hydrocarbon source comprises chemical pretreatment, heatpretreatment, oxidation of the hydrocarbon source, or a combinationthereof.

In an embodiment, solubilizing the disintegrated hydrocarbon sourcecomprises treating the disintegrated hydrocarbon source with at leastone enzyme. In an embodiment, admixing biochemical liquor comprisesadmixing at least one additional enzyme. In an embodiment, admixing atleast one additional enzyme further comprises admixing an enzyme forconverting a hydrocarbon source to lower molecular weight hydrocarbons.

In an embodiment, separating liquid hydrocarbons comprises a process ofsettling the slurry from the liquid hydrocarbon. In an embodiment,separating liquid hydrocarbons comprises settling contaminants from theliquid hydrocarbon. In an embodiment, enriching the liquid hydrocarboncomprises admixing the liquid hydrocarbon with at least one enzyme.

In an embodiment, the biochemical liquor comprises a modified enzyme. Inan embodiment, the modified enzyme comprises an enzyme that isgenetically modified. In an embodiment, the modified enzyme comprises anenzyme that is chemically modified.

In an embodiment, the method is conducted in-situ in a coal mine orex-situ on mined coal. In an embodiment, enriching the liquidhydrocarbons comprises improving the liquid hydrocarbon productqualities prior to distillation. In an embodiment, the liquidhydrocarbon product comprises at least one selected from the groupconsisting of gasoline, diesel, kerosene, and distillates thereof. In anembodiment, the hydrocarbon source comprises at least one selected fromthe group consisting of coal, anthracite coal, bituminous coal, lignite,sub-bituminous coal, low-rank coals, coke, tar sand, and oil shale.

Herein also disclosed is a method for in-situ coal to liquid hydrocarbonconversion, comprising: locating an underground coal seam; drilling atleast one well, the well in contact with the underground coal seam andhaving a means to cycle liquids therethrough; pressurizing theunderground coal seam with steam; cycling reactants through theunderground coal seam, wherein the reactants comprise at least oneenzyme, to form a slurry; withdrawing a portion of the slurry;processing the slurry to produce the liquid hydrocarbon; separating theliquid hydrocarbon from the slurry; and returning the slurry to the coalseam for further processing.

In an embodiment, the at least one well is in fluid communication with areactant stream. In an embodiment, the at least one well is in fluidcommunication with a slurry processing stream.

In an embodiment, cycling reactants to form a slurry further comprisessolubilizing the coal to form a slurry; converting the coal to formliquid hydrocarbons; separating contaminant compounds from the liquidhydrocarbons, wherein the contaminant compounds comprise pollutants;settling the slurry from the liquid hydrocarbons, wherein the liquidhydrocarbons are suitable for liquid fuels; and processing the liquidhydrocarbons to liquid fuels.

In an embodiment, the step of solubilizing the coal comprises treatingthe coal with at least one enzyme. In an embodiment, the step ofconverting the coal comprises treating the coal with at least oneenzyme. In an embodiment, the step of separating contaminant compoundscomprises treating the liquid hydrocarbons with at least one enzyme.

Further disclosed is a method for using an enzyme to produce liquidfuels, comprising selecting a microorganism, the microorganism producingan enzyme; modifying a microorganism genetically, to increase theproduction of the enzyme; modifying the enzyme structurally, to alterthe activity of the enzyme, to form a modified enzyme; collecting themodified enzyme, to form a biochemical liquor comprising at least onemodified enzyme; and exposing a hydrocarbon source to the biochemicalliquor to form a liquid fuel precursor.

In an embodiment, the step of selecting a microorganism comprisesselecting at least one microorganism chosen from the group consisting ofhypoliths, endoliths, cryptoliths, acidophiles, alkaliphiles,thermophiles, ithoautotrophs, halophiles, piezophiles, and combinationsthereof. In an embodiment, modifying a microorganism comprises insertinga nucleic acid vector. In an embodiment, modifying a microorganismgenetically comprises directed mutagenesis. In an embodiment, modifyingan enzyme comprises structurally changing an enzyme. In an embodiment,exposing the biochemical liquor to the hydrocarbon source furthercomprises transmitted-radiation directed fragmentation.

In one embodiment, a method for producing liquid hydrocarbon products,comprising, disintegrating a hydrocarbon source, treating thedisintegrated hydrocarbon source chemically, solubilizing thedisintegrated hydrocarbon source, admixing a biochemical liquor, whereinthe biochemical liquor comprises at least one enzyme to form liquidhydrocarbons, separating liquid hydrocarbons, and enriching the liquidhydrocarbons to form a liquid hydrocarbon product.

In another embodiment, a method for in-situ coal to liquid hydrocarbonconversion, comprising, locating an underground coal seam, drilling atleast one well, the well in contact with the underground coal seam;pressurizing the underground coal seam with steam; cycling reactantsthrough the underground coal seam, wherein the reactants comprise atleast one enzyme, to form a slurry; withdrawing a portion of the slurry;processing the slurry, wherein the liquid hydrocarbon is separated fromthe slurry; and returning the slurry to the coal seam for furtherprocessing.

In further embodiments, a method for using an enzyme to produce liquidfuels, comprising selecting a microorganism, the microorganism producingan enzyme; modifying a microorganism genetically, to increase theproduction of the enzyme; modifying the enzyme structurally, to alterthe activity of the enzyme, to form a modified enzyme; collecting themodified enzyme to form a biochemical liquor comprising at least onemodified enzyme; and exposing a hydrocarbon source to the biochemicalliquor to form a liquid fuel precursor.

The foregoing has outlined rather broadly the features and technicaladvantages of the invention in order that the detailed description ofthe invention that follows may be better understood. The variouscharacteristics described above, as well as other features, will bereadily apparent to those skilled in the art upon reading the followingdetailed description of the preferred embodiments, and by referring tothe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

For a detailed description of the preferred embodiments of theinvention, reference will now be made to the accompanying drawings ifvarious embodiments of the invention, in which:

FIG. 1 illustrates a general flow process schematic for converting coalto liquid.

FIG. 2 illustrates one embodiment of an ex-situ process for convertingcoal to liquid.

FIG. 3 illustrates one embodiment of an in-situ process for convertingcoal to liquid.

FIG. 4 illustrates a representative diagram of a catalytic antibody.

FIG. 5 illustrates a representative diagram of an activated catalyticantibody.

FIG. 6 illustrates a representative diagram of a wild type enzyme.

FIG. 7 illustrates a representative diagram of an activated enzymecomplex.

FIG. 8 illustrates a representative diagram of a directed evolution ofan activated enzyme complex.

FIG. 9 illustrates a representative diagram of site directed mutagenesisof an activated enzyme complex.

FIG. 10 illustrates a representative diagram of allosteric directedmutagenesis of an activated enzyme complex.

FIG. 11 illustrates a representative diagram of an active site redesignof an activated enzyme complex.

FIG. 12 illustrates a representative diagram of an active site rationaldesign of an activated enzyme complex.

FIG. 13 illustrates a representative diagram of a cofactor directedactive site redesign of an activated enzyme complex.

FIG. 14 illustrates a schematic of photofragmentation.

FIG. 15 illustrates a schematic of laser mediated photofragmentation.

FIG. 16 shows the results from the mechanical grinding run performed onthe Fluid Energy Model 0101 JET-O-MIZER 630 size reduction mill.

FIG. 17 shows the results obtained from the completed CFS Mill.

FIGS. 18A-18C are micrographs of product in water; 18A at magnificationof 125×; 18B at magnification of 430×; 18C at magnification of 1000×.

FIG. 19A illustrates the results from the 3% H2O2 pretreated coal inenzymatic solubilization and conversion.

FIG. 19B illustrates the results from the 3% H2O2 pretreated coal inchemical solubilization and conversion.

FIG. 19C illustrates the results from the 3% H2O2 pretreated coal in PDgrowth medium containing P. chrysosporium.

FIG. 19D illustrates the results from the 3% H2O2 pretreated coal in0.1× SD growth medium containing P. chrysosporium.

FIG. 19E illustrates the results from the 15% H2O2 pretreated coal inenzymatic solubilization and conversion.

FIG. 19F illustrates the results from the 15% H2O2 pretreated coal inchemical solubilization and conversion.

FIG. 19G illustrates the results from the 15% H2O2 pretreated coal in PDgrowth medium containing P. chrysosporium.

FIG. 19H illustrates the results from the 15% H2O2 pretreated coal in0.1× SD growth medium containing P. chrysosporium.

FIG. 19I illustrates the results from the 30% H2O2 pretreated coal inenzymatic solubilization and conversion.

FIG. 19J illustrates the results from the 30% H2O2 pretreated coal inchemical solubilization and conversion.

FIG. 19K illustrates the results from the 30% H2O2 pretreated coal in PDgrowth medium containing P. chrysosporium.

FIG. 19L illustrates the results from the 30% H2O2 pretreated coal in0.1× SD growth medium containing P. chrysosporium.

FIG. 19M illustrates the results from the heated, 30% H2O2 pretreatedcoal in enzymatic solubilization and conversion.

FIG. 20A illustrates the results from the heated coal in PD growthmedium containing P. chrysosporium.

FIG. 20B illustrates the results from the heated coal in 0.1× SD growthmedium containing P. chrysosporium.

FIG. 21A illustrates the results from the mine water pretreated coal inenzymatic solubilization and conversion.

FIG. 21B illustrates the results from the mine water pretreated coal inchemical solubilization and conversion.

FIG. 21C illustrates the results from the mine water pretreated coal inPD growth medium containing P. chrysosporium.

FIG. 21D illustrates the results from the mine water pretreated coal in0.1× SD growth medium containing P. chrysosporium.

FIG. 22A illustrates the results from the HNO3 (pH 1) pretreated coal inenzymatic solubilization and conversion.

FIG. 22B illustrates the results from the HNO3 (pH 1) pretreated coal inPD growth medium containing P. chrysosporium.

FIG. 22C illustrates the results from the HNO3 (pH 1) pretreated coal in0.1× SD growth medium containing P. chrysosporium.

FIG. 23 (prior art) is a simplified diagram showing microbialdegradation of phenol.

FIG. 24 (prior art) illustrates UV spectra for phenol degradation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A process for converting coal to liquid fuels is disclosed. Coalcomprises any coal found or removed from a coal mine, seam, or pit. Thecoal may further comprise anthracite coals, or the coke from bituminouscoal. In certain instances, the coal comprises lignite, sub-bituminouscoal, other low-rank coals, and/or other hydrocarbon source, such as tarsands, without limitation. Alternatively, the coal comprises weathered,aged, leached, or degraded coal without limitation.

In certain instances, the coal is converted to liquid fuels by atwo-stage process. The process comprises ezymatically-catalyzedreactions. The first stage, Stage I, comprises the pretreatment andconversion of coal into liquid products by enzymes. In embodiments,Stage I converts coal feedstocks to liquid hydrocarbons. The feedstockscomprise coal remaining in coal mines, or in-situ feedstocks.Alternatively, the feedstocks comprise coal away from the coal mine, orex-situ feedstocks. In certain instances, the source of hydrocarbons maycomprise any source, such as tar sands, not preferred for otherindustries.

The second stage, Stage II, comprises the enrichment of the liquidhydrocarbon products. The enrichment, or improvement, of the liquidhydrocarbon product comprises further enzymatically-catalyzed reactions.Additionally, the reactions are enzyme-mediated processing steps. StageII further comprises the improvement of the liquid product propertiesfor use in fuels. The Stage II enzymatically-catalyzed processes changethe fuel performance. In exemplary instances, Stage II processing mayalter the cetane rating of diesel, or the octane rating of gasoline.

Compared to current processing techniques, the enzymatically-mediatedtwo-stage process requires less energy for processing. Additionally, thereaction conditions are milder, as the enzymes perform optimally inhomeostatic conditions of the microorganism producer. Further,additional enzymes or microorganisms may be implemented to sequestercontaminants regulated in liquid fuels. For instance, sulfur andnitrogen may be reduced or removed from the liquid fuels prior to finalproduct distillation. The early removal of polluting contaminants makesthe process adjustable to meet current and future emissions regulations.Additionally, the enzyme-mediated two-stage process is adaptable tofeedstocks previously inaccessible, for instance, coal columns in a minetoo dangerous for removal by retreat mining.

Microorganisms. In the disclosed process, microorganism-produced enzymesmediate the conversion. In embodiments, the microorganisms comprisebacteria, algae, or fungi. In certain instances, the microorganismscomprise heterotrophs that secrete enzymes for catalytic digestion ofhydrocarbons. The microorganisms use hydrocarbons as a carbon source forlife processes. The microorganisms are harvested from oil shales, oilsands, coal tar pits, or coal caves without limitation. For example, themicroorganisms may be derived from those found in the La Brea Tar Pits.Further, the microorganisms may be collected from geothermal springs,mud volcanoes, sulfur cauldrons, fumaroles, geysers, mudpots, or thelike, without limitation. The microorganisms may further compriseextremophiles, such as but not limited to hypoliths, endoliths,cryptoliths, acidophiles, alkaliphiles, thermophiles, ithoautotrophs,halophiles, or piezophiles. In exemplary embodiments, the microorganismscomprise archaebacteria. Alternatively, suitable microorganisms includethose found in the geneses Poria, Polyporus, Thiobacillus, Candida,Streptomyces, Psuedomonas, Penicillium, or Trichoderma. As understood byone skilled in the art, alternative microorganisms may be identifiedthat are suitable for application in the disclosed system, without beingnamed specifically, that do not vary in structure and functionsignificantly. Further, it can be envisioned that these microorganismsare envisioned as means to alter, improve, or modify the currentdisclosure.

In certain instances, the microorganisms are exposed to previously minedcoal, coal residues, or coal residues less favorable for powerproduction in order to harvest the enzymes. In instances, themicroorganism produces the enzymes naturally. As understood by oneskilled in the art, continued exposure to the substrate, such as coal,will lead to increased expression and production of the enzymes for theliquid hydrocarbon production.

Enzymes. The enzymes used in the disclosed process are obtained frommicroorganisms that produce these enzymes in high yields. In furtherembodiments, the microorganisms are genetically altered to produce theenzymes in high yields. Preferably, the enzymes are secretedextracellularly and/or release the enzymes into their environment.Alternatively, the cells are lysed and the enzymes are captured for usein coal processing. In embodiments, the enzymes are separated from themicroorganisms prior to use in the processing of coal. In order toreduce exposure, release, or environmental contamination, themicroorganisms are separated from the processing. No microorganisms aredirectly involved in any embodiments of the liquid fuel productionprocess. Furthermore, the facilities used to grow these organisms havesufficient provisions to isolate host organisms from the naturalenvironment.

Alternatively, the microorganisms undergo site-directed mutagenesis toup-regulate, over-express, and/or increase, the production of enzymes.Site directed mutagenesis comprises the mutation of a DNA molecule atspecific nucleic-acid base-pair sequence. Site directed mutagenesis mayoccur in the chromosomal DNA, or in extra-chromosomal DNA, from avector. Additionally, the site-directed mutagenesis may comprise genedeletion/excision, primer mediated mutagenesis, cassette mutagenesis,add-on mutagenesis, mismatch mutagenesis, gene conversion, topologicalmanipulation, specialized recombination, or PCR-mediated mutagenesis.Further, mutagenesis and over-expression of a gene may be induced by anymutagen. For instance, ionizing radiation, UV exposure, deamination,intercalation, alkylation, analog insertion, transposon multiplication,and other molecular biology techniques may be used, without limitation.In certain instances, mutagen exposure induces the microorganism toacquire a vector, such as a plasmid. Further, mutagenesis may induce theincorporation of vector DNA into the chromosomal DNA. For the purpose ofthis disclosure, directed, mutagenic technique may be implemented inorder to induce additional production of an enzyme. Further, themutagenesis may be used to increase the activity of the enzyme.

In certain instances, the enzymes are chemically modified afterproduction. The enzymes may be modified prior to or after harvestingfrom the microorganisms. Any process known to one skilled in the art maybe implemented, such as but not limited to addition of functionalgroups, addition of other peptides, altering the chemical nature, and/orstructural changes. For example, processes like acylation,glycosylation, ubiquitination, deamidation, and/or cleavage, areenvisioned to have applications within the present disclosure, withoutlimitation.

The produced and modified enzymes are utilized in biochemical liquor.The biochemical liquor comprises a liquid mixture of proteins, enzymes,inorganic catalysts, and organic and inorganic compounds. Thebiochemical liquor further comprises salts, electrolytes, metals, and/orother molecules that aid, improve, or alter the operations of an enzymebroth, or biochemical liquor. The biochemical liquor is a mixture of atleast one of each of the aforementioned groups without limitation. Thebiochemical broth may be suspended in any known solvent; preferablyorganic solvents. In exemplary embodiments, water is the solvent.

Process. As illustrated in FIG. 1, the process comprises a flow of coalthrough a sequence of individual treatment steps. In STAGE I, theprocess comprises at least one pretreatment step. The coal thenundergoes solubilization, which may be included in the pretreatmentsteps. The pretreated, solubilized coal material is converted to liquidhydrocarbons. Simultaneously, or sequentially, the material may undergosulfur and nitrogen conversion. Removing sulfur and nitrogen from theproduct improves performance of the fuel after processing, such asseparation. The different fuels are separated, such that aqueous phasereactants are recycled, and/or the wastewater is treated for return tothe system. In STAGE II, the hydrocarbon phase products from theconversion step are further refined. The refining of the productscomprises fuel refining, enrichment, and distillation to improve productqualities.

Pretreatment. The pretreatment step comprises a physical and chemicaldegradation of the coal, to produce a degraded coal. In order toincrease efficiency of the disclosed process it is advantageous toincrease the surface area of the material. The surface area of the coalmay be increased by reducing the particle volume, such as in the processof comminution. For instance, surface coal, mined coal, coal tailings,or remaindered coal is mechanically broken down or crushed into a fineparticulate. Remaindered coal may comprise, without limitation, coalsourced from any industry from which it was rejected for use. Theparticulate may comprise pebbles, dusts, powders, or the like withoutlimitation.

In certain instances, the coal is treated in-situ, for instance, in anunderground coal seam, by high-pressure steam. Underground wells,conduits, and/or other lines deliver steam to a coal mine. The steam maybe pressurized, superheated, or combinations thereof in order toincrease penetration into the seam. The steam is used to fracture thecoal bed underground prior to pretreatment. Further, the high-pressuresteam mechanically separates the coal from surrounding rock formations.

Additionally, the coal is subjected to a chemical treatment. Chemicaltreatment of the coal increases the reactivity of the coal.Additionally, chemical treatment is designed to remove, digest, oreliminate non-coal materials from the coal products. Further, thechemical treatment may increase the surface area of the coal further byinducing the formation of pores, cavities, pits, and the like. Thechemical treatment may compromise an oxidative agent. In certainembodiments, mild ionic, acid, base, or free radical solutions areapplied to oxidize the coal. In an embodiment, the solution comprises amild acid solution. In an exemplary embodiment, the chemical treatmentcomprises hydrogen peroxide. The hydrogen peroxide is in concentrationsbetween about 10% and about 50%; alternatively between about 20% andabout 40%, and in preferably about 30% hydrogen peroxide. As discussedabove, the solutions are injected into coal mines with steam, or water,therefore it is preferable the solutions are aqueous. In certaininstances, an inorganic chemical treatment may be implemented.

Solubilization. Further, the pretreatment steps comprise solubilizationof the degraded coal. Once mechanically disintegrated, and chemicallyoxidized, a first set of enzymes is introduced to break thecross-linking bonds in coal. In certain instances, the first enzymes maybe derived from enzymes found, for example, in the genera Peoria,Polypore's, genuses Poria, Polyporus, without limitation. The firstenzymes allow the coal particles to dissolve into the liquid medium. Inembodiments, the liquid medium is the same as is used to deliver theenzymes. In certain instances, the medium is the biochemical liquordescribed above. Further, the liquid medium comprises an aqueous medium.The solubilized coal particles are suspended in the liquid mediumforming a coal slurry; alternatively, a coal suspension, a coal mixture,a coal colloid, or a coal solution, without limitation. The coal slurryimproves accessibility to the coal particles by the enzymes from thebiochemical liquor. Suspending the particles in the medium may improvereaction kinetics during the subsequent enzymatically mediated steps.The coal slurry further improves transfer of the coal between processingsteps.

Conversion. After solubilization, the coal is processed by conversionsteps. In certain instances, the solubilized coal in the coal slurry isconverted to smaller or lower hydrocarbons. The lower hydrocarbons maycomprise any hydrocarbon, for instance hydrocarbons comprising betweenabout 24 carbons and about 2 carbons. The second enzyme, or secondenzyme solution, is maintained in the biochemical broth. The secondenzyme solution may be derived from enzymes produced by microorganismsfor the genera, Thiobacillus, Candida, Streptomyces, Psuedomonas,Penicillium, Trichoderma, for example, without limitation. The secondenzyme may be introduced to the coal slurry during the conversionprocess. In certain instances, the exposure of the coal slurry to asecond enzyme comprises converting coal to lower molecular weighthydrocarbon fractions. Alternatively, the second enzyme is a convertingenzyme. Converting enzymes are those selected, engineered, or modifiedto catalytically convert large hydrocarbon molecules found in coal tolower molecular weight hydrocarbons. During the conversion step, thehydrocarbons undergo saturation, and sulfur, nitrogen, and othercontaminant conversion. The conversion step forms a reaction slurry, ora hydrocarbon slurry with the biochemical liquor.

The enzymatic conversion reactions successively break the native,original, or solubilized, coal particles into smaller hydrocarbonmolecules that remain in the reaction slurry. The enzymatic conversionreactions convert the high molecular weight molecular components of coalto lower molecular weight mixtures of hydrocarbon liquids andhydrocarbon gases.

During conversion, certain waste products, contaminants, and potentialpollutants are removed from the process. In certain instances, theremoval of these products is mediated by a third enzyme added to thereaction slurry. The third enzyme, or third enzyme solution, reacts withthe products of catalytic conversion to liberate sulfur from thehydrocarbon complexes and form a variety of simpler sulfur-containingcompounds, which are soluble in the reaction slurry. The solublesulfur-containing compounds may be filtered from the reaction slurry andprocessed for other products.

Additionally, in order to remove other waste products, contaminants, andpotential pollutants, a fourth enzyme may be added to the reactionslurry. In certain instances, any number of waste removal enzymes may beused to specifically eliminate, sequester, or cleave the unwantedcompounds. In certain embodiments, the fourth enzyme solution reactswith the products of catalytic conversion to liberate nitrogen and forma variety of simpler nitrogen-containing compounds, which are soluble inreaction slurry.

The processed wastes may comprise gases solubilized in the reactionslurry. Gases may comprise nitrogen, oxides of nitrogen, sulfur, oxidesof sulfur, carbon monoxide, carbon dioxide, and other gases withoutlimitation. Certain waste products are used for further processes, suchas syngas production, or catalyzed synthesis of liquid fuels. Further,enzymatically-catalyzed reactions convert the complex sulfur andnitrogen compounds found in coal to simpler forms that are removedduring product separation.

As understood by one skilled in the art, the reaction properties such astemperature, pressure, pH and residence time are differentiallymonitored, and controlled for maximized production. In certaininstances, the reaction properties are controlled to obtain adistribution of hydrocarbon molecular weights in the product stream.Further, as the reaction slurry comprises the biochemical liquor,altering the conditions may optimize conversion.

As discussed hereinabove, the biochemical liquor comprises any number ofenzymes. As understood by one skilled in the art, each enzyme haspreferred conditions for efficient catalysis. As such, cycling thereaction conditions, such as temperature and pressure, is envisioned tomaximize the efficiency of any portion of the process, or the action ofany portion of the enzymes. In other embodiments, the conversion stepconsists of separate reaction vessels for the solubilization, catalyticcracking, and nitrogen and sulfur conversion reactions. Such anarrangement permits different operating conditions to be used in eachvessel, such as temperature and individual reactor recycle rates, tooptimize the enzyme-catalyzed reactions.

Product Separation. Following conversion, the hydrocarbon mixture issent into settling tanks. In embodiments, the settling tank may be anyvessel configured for separating the hydrocarbon liquid from the aqueouscoal slurry. In certain instances, the settling tank may comprise adynamic settler, wherein a constant low volume, or slow velocity, streamof the reaction slurry is introduced to separate the aqueous andhydrocarbon phases. Alternatively, the settling tank comprises a staticsettler, where the aqueous phase coal slurry settles from the lighterhydrocarbons by virtue of gravity. In certain embodiments, the remainingsulfur and nitrogen compounds distribute to the water phase. Thehydrocarbon phase is separated by drawing off the lighter hydrocarbonlayer from the denser aqueous layer. Alternatively, a conduit withdrawsthe aqueous phase from the bottom of the tank.

Product Enrichment. The hydrocarbon layer, which already containsgasoline, kerosene, diesel, and fuel oils is sent to Stage II where itis further upgraded by converting the lower-valued fractions, naphtha,diesel, fuel oils, waxes, and the like to higher-valued fractions suchas gasoline or kerosene. In embodiments, the product enrichment maycomprise enzymatic conversion, molecular photofragmentation, conversion,and enzyme assisted molecular photo-fragmentation conversion. In certaininstances, the product enrichment comprises a fifth enzyme, or fifthenzyme solution introduced to the hydrocarbon products from theseparation step. Following enrichment, the hydrocarbon mixture isseparated into final products by conventional distillation.

Ex-situ Processing. FIG. 2 illustrates an embodiment of processing ofex-situ coal feedstocks continuously, or an EX-system 10. In theEX-system 10, pretreatment, conversion, and product processes aredesigned to be in fluid communication. In EX-system 10, the coal isfirst ground into small particles by mechanical means 12. As previouslydescribed, the mechanical means 12 creates a particulate product stream14. The particulate product stream is introduced to chemical treatmentsystem 16, comprising, for example, a weak acid solution. In certaininstances, mechanical means 12 and chemical treatment system 16 maycomprise a single vessel, or single processing facility. The coal andacid solution form coal slurry where the coal undergoes pre-oxidation.The extent of the pre-oxidation is determined by the residence time ofthe slurry in the chemical treatment system 16. Further, in combinedembodiments, the oxidation of the coal in the coal slurry may becontrolled, at least in part, by the degree of agitation provided bymixers 18.

The slurry product stream 20 is then pumped out of the feed tank andinto a reactor stage 22. The reactor stage 22 comprises thesolubilization reaction. In certain instances, the first enzyme stream24, with enzymes selected for solubilizing the coal, is injected intoslurry product stream 20. Alternatively, first enzyme stream 24 isinjected directly into reactor stage 22. Without wishing to be limitedby theory, it may be beneficial for first enzyme stream 24 to beintroduced to slurry stream 20 prior to introduction to reactor stage22.

Reactor stage 22 comprises the enzyme mediated catalytic conversionreaction. Second enzyme stream 26 is injected into reactor stage 22. Theenzymes react catalytically, convert the large hydrocarbon molecules,and produce product stream 30. Further, third enzyme stream 27 toconvert sulfur compounds and fourth enzyme stream 28 to convert thenitrogen compounds are added to reactor stage 22. The third enzymestream 27 and fourth enzyme stream 28 convert the respectivecontaminants found in the coal slurry 20 into simpler, water-solubleforms. Reactor stage effluent 23 is continuously split between a recyclestream 25, which is pumped back into the reactor stage 22, and a productstream 30.

In other embodiments, the reaction stage 22 consists of separatereaction vessels for the solubilization, catalytic conversion, andnitrogen and sulfur conversion reactions. A multiple reactor arrangementpermits different operating conditions to be used in each vessel, suchas temperature and individual reactor recycle rates, to optimize thedifferent suites of reactions.

The product stream 30 is pumped into the separation stage 32. Separationstage 32 may comprise gas vent 33 for withdrawing the gases and volatilecompounds released during separation. In certain instances, gas vent 33vents some gases that were dissolved in product stream 30. Theseparation stage 32 comprises the step where the aqueous phase 36settles out under the hydrocarbon phase 34. Separation stage 32comprises a settler, or settling vessel. Alternatively, separation stageis a filter or other apparatus to separate aqueous and hydrocarbonphases from product stream 30. Separation stage 32 comprises acontinuous flow, oil separation vessel. The aqueous phase 36 iswithdrawn from separation stage 32 and routed via recycle stream 39 tothe reactor stage 22. The recycle stream comprises a wastewatertreatment system. The treatment system comprises any system configuredas a sour water treatment, configured to remove residuum, as well asnitrogen and sulfur by-products. Treated water is then recycled back tothe reactor section.

In certain instances, the hydrocarbon phase 34 may be withdrawn from thetop of the settler as hydrocarbon stream 38 for enrichment and/ordistillation to produce transportation fuels. The hydrocarbon stream 38is pumped to a product enrichment stage.

In-Situ Processing. Another embodiment involves the continuousprocessing of in-situ coal, or IN-system 100, which is illustrated inFIG. 3. In this embodiment, the IN-system 100 comprises an abandoned,collapsed, inaccessible, or otherwise difficult to mine coal deposit, orunderground coal seam 101. In embodiments, at least one well 102 isdrilled into the coal seam 101. The wells 102 are generally configuredfor the transport of liquids and slurries between the underground coalseam 101 and the processing center 120. Further, the wells 102 may beconfigured for the continuous circulation of process fluids in and outof the underground coal seam 101. It can be envisioned that a pluralityof wells 102 would improve product yield, processing time, and thegeneral economics of the IN-system 101, without limitation.

The IN-system 100 process begins with the injection of steam in the well102 conduits to induce fracturing of the underground coal seam 101. Thisfracturing step 105 is configured to break the coal seam intoparticulates, coal gravel, or the like. In certain instances, usinghigh-pressure steam is according to conventional practices of theunderground coal mining industry.

After the fracturing step 105 is complete, the high-pressure steam iswithdrawn. The oxidation step 106 comprises injecting an acid solutionto pre-oxidize the fractured coal. In embodiments, the acid solution isrecycled continuously to form a circulating process stream 150, suchthat the acid is pumped into well 102A at one side of the seam, pumpedout of well 102B on the other side of the seam. To complete the cycle,the acid solution is transported back and pumped into the first well102A. The circulating process stream 150 may be repeated until thedesired level of pre-oxidation is achieved.

As described above, the solubilization step 107 may comprise theintroduction of the first enzyme solution into the underground coal seam101. The first enzyme solution is introduced into the circulatingprocess stream 150 to solubilize the exposed coal into the circulatingprocess solution.

Once an adequate level of soluble coal is achieved, to create coalslurry in the circulating process stream 150, the additional enzymes areadded either sequentially or simultaneously. In certain instances, thesecond enzyme 106, third enzyme 107, and fourth enzyme 108 solutions areadded to the circulating stream 150. As described herein above, thesecond enzyme stream 106 is selected to catalytically crack thehydrocarbons. The third enzyme 107 and fourth enzyme 108 solutions areselected to remove sulfur and nitrogen-containing compounds and/or wasteproducts from the circulating process stream 150. In embodiments,further enzyme streams may be injected into circulating process stream150 to optimize the solubilization and conversion of the coal.

A portion of the circulating process stream 150 is then split and takenas a raw product stream 160 and sent to a processing stage 120. Theprocessing stage 120 is similar to the one used in ex-situ embodimentsof the process described above. Separation vessel 162 comprises the stepwhere the aqueous phase 163 settles out under the hydrocarbon phase 164.Separation vessel 162 comprises a settler or settling vessel. Theaqueous phase 163 is withdrawn from separation vessel 162 and routed viarecycle stream 170 to the circulating process stream 150. The recyclestream 170 comprises a wastewater treatment system. The treatment systemcomprises any system configured for sour water treatment, configured toremove residuum, as well as nitrogen and sulfur by-products. In certaininstances, the hydrocarbon phase 164 may be withdrawn from the top ofthe settler as hydrocarbon stream 168 for enrichment and/or distillationto produce transportation fuels. The hydrocarbon stream 38 is pumped toa product enrichment stage.

Mutagenesis. Methods used to produce suitable enzymes for implementationin Stage II for fuel upgrade include using catalytic antibodies. Asillustrated in FIG. 4, biological enzymes are identified for thecatalytic processes desired. In certain instances, the biologicalenzymes are derived from the microorganisms discussed herein above. Infurther embodiments, the enzymes comprise Mother Nature only (MNO)enzymes. MNO enzymes are the phenotypic expressions of unmodifiedgenetic sequences within the microorganisms. Alternatively, MNO enzymesare wild-type enzymes. In further instances, illustrated in FIG. 5, theMNO enzymes are selected from those that comprise an activated enzyme.In certain instances, the activated enzyme screening is conducted by anantibody assay. Alternatively, any suitable screening method maycomprise any suitable protocol to identify the wild type MNO enzymes, asfurther illustrated in FIGS. 6 and 7.

The MNOs selected are formed by directed evolution, as illustrated inFIG. 8. The selected MNOs are subject to site-directed and randommutagenesis throughout the enzyme, not solely restricted to the activesite. In certain instances, the enzymes are also subject to mutagenesisat allosteric sites, and at sites remote from active and/or allostericsites. The mutagenesis at multiple sites comprises a means to bothpromote and restrict potential products as illustrated in FIGS. 9 and10. In certain instances, the mutagenesis includes active site chemicalredesign as shown in FIG. 11. Preferably, the results include a rationaldesign enzyme, or enzymatic structure.

The structure is synthesized, computationally designed, with motifsattached to enzyme scaffolds. As enzymes are rather large molecules,having hundreds of amino acids, tens of kilo Daltons (Kds), andthousands of cubic angstroms, they may be considered spatiallyinefficient. In certain instances, large enzyme molecules comprise smallactive sites. Enzymatic reactive sites are quite small by comparison andthe other folded amino acids serve as a scaffolding to create thereactive site volume. These “other” amino acids can be, relativelyspeaking, quite far from the active site of the enzyme as illustrated inFIG. 12. Additionally, the enzymes may include cofactor attachment siteredesigns, shown in FIG. 13. In order to induce cofactor attachment siteredesigns the implementation of site directed mutagenesis is repeated asdiscussed hereinabove, for example, paragraph 21.

As diagrammed in FIG. 14, a shaped IR femtosecond laser pulse may beimpinged upon the enzymatic complex to induce reactant fragmentation.Further, it can be envisioned that any particular impingent radiation,known to one skilled in the art, may be capable of the same reactantfragmentation, without limitation. The laser pulse for directedfragmentation of reactants/conversion to products may aid the formationof reactant products. As understood by one skilled in the art, multiplefragmentation reactant products may be formed. In certain instances, themultiple fragmentation products may be advantageous for the formation ofa range of reactant fragments. Alternatively, the shaped IR femtosecondlaser pulses in conjunction with above mentioned enzyme techniquesassist in selective fragmentation of reactants at enzymatic activesites, allosteric sites, and sites remote from binding or allostericsites as shown in FIG. 15. As understood by one skilled in the art, thebonding of the reactant, hydrocarbon, molecular to the enzyme reactivesite may comprise a covalent, non-covalent, hydrogen, ionic, Van derWaals, or other bond, interaction, coupling, or association, withoutlimitation. Further, the enzyme reactive site is configured to restrictthe reactant molecule, and its range of movement. Further, the reactivesite restricts internal degrees of freedom, to favorably target thefemtosecond laser pulses to the preselected internal bond. In certaininstances, the enzyme reactive site damps the internal degrees offreedom, such that internal vibrational rearrangement (IVR) isprevented, and the laser energy is focused to the preselected internalbond.

Conversion of coal to liquid hydrocarbons. In an embodiment, a processof converting coal to liquid hydrocarbons comprises mechanicalpretreatment and/or chemical pretreatment, solubilization, andconversion (from coal to liquid hydrocarbons). Mechanical pretreatmentincludes hammer mill grinding or jet mill grinding. In some cases, jetmill grinding is able to grind coal into particles with sizes of 5 μm orless. For example, a Fluid Energy Model 0101 JET-O-MIZER-630 sizereduction mill may be used.

In an embodiment, solubilization and conversion are performed on variouspretreated coal. In some cases, coal is pretreated by an enzymaticprocess, for example, using extracellular Laccase and ManganesePeroxidase (MnP). In some cases, coal is pretreated by a chemicalprocess, for example, using Ammonium Tartrate and Manganese Peroxidase.In some cases, coal is pretreated by an enzymatic process, for example,using live organisms Phanerochaete chrysosporium.

EXAMPLE

Overview. Coal is decomposed with three different approaches (1) anenzymatic process—using extracellular Laccase and Manganese Peroxidase(MnP); (2) a chemical process—using Ammonium Tartrate and ManganesePeroxidase; and (3) an enzymatic process—using live organismsPhanerochaete chrysosporium. Spectral analysis was used to determine howeffective each of these methods is in decomposing bituminous coal. Afteranalysis of the results and other considerations, such as cost andenvironmental impacts, it was determined that the enzymatic approaches,as opposed to the chemical approaches using chelators, were moreeffective in decomposing coal. The results from the laccase/MnPexperiments and Phanerochaete chrysosporium experiments are presentedand compared.

Spectra from both enzymatic methods show absorption peaks in the 240 nmto 300 nm region. These peaks correspond to aromatic intermediatesformed when breaking down the coal structure. The peaks then decrease inabsorbance over time, corresponding to the consumption of aromaticintermediates as they undergo ring cleavage. The results show that thisprocess happens within 1 hour when using extracellular enzymes, buttakes several days when using live organisms. In addition, liveorganisms require specific culture conditions, control of contaminantsand fungicides in order to effectively produce extracellular enzymesthat degrade coal. Therefore, when comparing the two enzymatic methods,results show that the process of using extracellular lignin degradingenzymes, such as laccase and manganese peroxidase, appears to be a moreefficient method of decomposing bituminous coal.

Mechanical Pretreatment. The mechanical pretreatment process involvesgrinding the coal particles down to a smaller size before chemicalpretreatment and experimentation. This process provides an increasedsurface area for both the chemical pretreatment of coal as well as agreater surface area for the subsequent enzymatic conversion of coal toliquid hydrocarbons.

Bituminous coal was used, Lower Kittanning Seam, high vol—No. 5, fromRosebud Mining Company, Kittanning, Pa.

In Q1, three different coal grinds were used: 1 mm, 400 μm and 40 μmsized particles. All three of these grinds were performed using hammermill grinding. The 1 mm particles came from Rosebud Coal, Kittanning,Pa. The 400 μm and 40 μm particles came from Pulva Corporation,Valencia, Pa. (although the starting material for the micron sizedgrinds was also 1 mm Rosebud coal). The mechanical pretreatment processin Q2 was changed from hammer mill grinding to jet mill grinding. Thiswas performed by Fluid Energy, Inc., Telford, Pa. The equipment used wasa JET-O-MIZER Size Reduction System. The actual grinding was done undersealed conditions, excluding any oxygen or air. This was done for safetyreasons, to guard against any spontaneous ignition or explosions.Further mechanical grinding work was performed in Q3 using a FluidEnergy reduction mill (e.g., Fluid Energy Model 0101 JET-O-MIZER 630size reduction mill). The Fluid Energy Model 0101 JET-O-MIZER 630 sizereduction mill takes 3 mm sized coal particles for input. This particlesize is readily available from coal producers. At a specific energyconsumption of roughly 1,000 kWh/t, using steam as the motive gas, meanparticle sizes of less than 5 microns are obtained.

Results and Discussion. FIG. 16 shows the results from the mechanicalgrinding run performed on the Fluid Energy Model 0101 JET-O-MIZER 630size reduction mill. As seen in this figure, 25% of the particles areless than 1.7 microns and 50% are less than 4 microns. The Sauter meandiameter, D(3,2), is only 2.128 microns and the specific surface area isover 28,000 cm2 per cm3. These small particle sizes result in anincreased surface area, which means an increase in the number offunctional groups of coal that become exposed to enzymes during thesolubilization and hydrocarbon conversion process (see Table 1 below).

Model 0101 JET-O-MIZER CFS Test Mill is abbreviated as “CFS Mill”, whichwas fitted with a steam interface to the grinder. The dimensions of theCFS Mill are roughly 7×6×3 feet. Behind the control panel on the rightare 2 heaters for steam. A coal hopper feeds coal into the grindingsection (jacketed cube to left of hopper). Ground coal product is fedinto a container behind the jacketed grinder.

TABLE 1 COULTER ® LS Particle Size Analyzer 13:25 Fluid Energy Ajet Filename: CL 10327.303 Group ID: CL 10027 Sample ID: COAL FUELS; COAL; RUN 3Run number: 79 Operator: YP Comments: PSI-100 N2; F/r-3M-R JCM0101Optical model: Fraunhofer LS 230 Small Volume Module Start Time: 13:21Run length: 60 seconds Observation: 7% PIDS obocur: 54% Fluid:2-Propanol Software: 2.09 Firmware 2.02 2.02

FIG. 17 shows the final results obtained from the completed CFS Mill.The coal product statistics are taken from a liquid product. Theseresults come from a MICROTRAC Standard Range Analyzer (SRA 150). As seenin FIG. 17, the grinding on the CFS Mill is done at a feed rate of coalof 27 pounds per hour. The motive steam is at 450° F. and 110 PSIG. Thecoal particle range is roughly from 0.5 microns to 25 microns. The meanvalue is 5.769 microns.

In addition to the statistical results, the coal from the CFS Mill wasexamined under a microscope. In the following micrographs, a number offeatures of the coal grind and particles are evident.

FIG. 18A is a micrograph of product in water. The magnification is 125×.The graticuled microscope slide is divided in tenths (0.1 mm) of amillimeter, as can be seen in the 0 to 0.5 divisions. The 0.1 mmdivisions are further subdivided again into tenths (0.01 mm) in the 0.0to 0.1 mm subdivision. The 0.01 mm divisions look like “guitar strings.”The large particle in the middle of the “guitar strings” is about 30microns in diameter. It is the largest particle in the visible field.Some particles are much, much smaller as can be seen in the next 2micrographs.

FIG. 18B is at magnification 430×. The guitar strings are divisions of10-5 m. As can be seen between strings and around the central field,there are particles 0.1 of the string divisions or about 1 micron. FIG.18C is a micrograph of the same central field as FIGS. 18A-18B. Themagnification is 1,000×. At this magnification, please note the orangecircled particle. It is about 10 microns in diameter. Naturally there isdiffraction of visible light around particles of this size. However,note that there appears to be some transparency through the middle ofthe particle. This phenomenon is also apparent throughout otherparticles on this slide as well as on other, not shown, micrographs.

A few different approaches for mechanical pretreatment were used in thisexample. Ultimately, it was decided to use jet mill grinding (as opposedto hammer mill grinding) which is able to grind the coal particles to 5μ size or smaller. As seen in the results, a Fluid Energy Model 0101JET-O-MIZER 630 size reduction mill is able to perform to the requiredspecifications and grind the coal particles to sizes 5 μm or less. Thecoal grinding process is extremely important because at particle sizesof 5 μm or less, the dramatically increased surface area providesadditional exposure of functional groups that then become exposed toenzymes. This is beneficial because increased reaction rates and productvolumes are highly dependent upon access, or exposure, to thesefunctional groups.

The particle sizes and dispersion of coal particles borders on thedefinition of “solubility.” The particles are on the order of 800-1,200benzene rings. These are quite favorable sizes to be “fed” to the seriesof enzymes to produce liquid fuels.

Solubilization and Conversion. Bituminous coal was used (LowerKittanning Seam, high vol—No. 5, from Rosebud Mining Company,Kittanning, Pa.). A mechanical grinder was used to grind the coal priorto running the experiments. As mentioned in the mechanical pretreatmentsection, a few different particle sizes were used but most of theexperiments were conducted using about 5 μm sized coal particles. Theresults analyzed are from the experiments using the 5 μm sized coalparticles.

Chemical pretreatment is used to “weather” or oxidize the coal, which inturn assists in the solubility of the coal in the enzymatic conversionprocess. Several different pretreatments were used in the experiments,including: hydrogen peroxide, H2O2 (3% pH 5, 15% pH 4.5, and 30% pH 4),PBS Mine H2O (Somerset, Pa. pH 2.2), nitric acid, HNO3 (pH 1, pH 2, pH3, pH 4, and 15M), distilled H2O, and preheated coal, in which the 5 μmcoal was preheated at a temperature of 120° C. for approximately 36hours. All of the pretreated samples were dried before the coal was usedfor the experiments.

In addition to conducting experiments with the different types ofpretreated coal, experiments were also conducted with untreated coal.The results from pretreated coal experiments were analyzed to determinethe most effective pretreatments. From these results, it was determinedthat the most effective pretreatments were hydrogen peroxide (3%, 15%,and 30%), heated coal, mine water, and nitric acid. The results from theexperiments using these specific pretreated coal samples are discussed.

Enzymatic Approach: Laccase and Manganese Peroxidase

Enzymatic experiments in this example were conducted using Laccase fromTrametes versicolor and Manganese Peroxidase (MnP) from Phanerochaetechrysosporium, both purchased from Sigma-Aldrich. Laccase and MnP areextracellular enzymes produced by white rot fungi. They are consideredto be two of the major groups of ligninolytic enzymes and are capable ofefficiently degrading lignin. Laccase was used for the first step of theprocess, enzymatic solubilization, and MnP was used in the second stepof the process, hydrocarbon conversion. The detailed experimentalprocedure for this approach is shown below.

Laccase and MnP Procedure

Step 1: Laccase Solutions:

-   -   Laccase buffer—100 mM citric acid-100 mM sodium phosphate        buffer, pH 4.5    -   Laccase solution—laccase (T. versicolor) was dissolved in        laccase buffer

Procedure

-   -   1) 0.1 g of dried, pretreated coal was combined with 3 ml of        laccase buffer in a test tube    -   2) 2 ml of laccase solution added to each test tube containing        the coal and laccase buffer    -   3) Each test tube was shaken on a Shaker Lab Line Orbital Shaker        overnight    -   4) Products from experiments were measured using the Genesys 10        UV-VIS spectrophotometer (range 190 nm-1100 nm), purchased from        Fisher Scientific. The product concentrations (number of drops)        measured in the spectrophotometer were adjusted so that the        absorption intensities were relatively the same.

Step 2: Manganese Peroxidase (MnP)

Solutions

-   -   MnP Buffer—160 mM malonic acid solution, pH 4.5    -   MnP solution—MnP oxidizer solution was mixed in MnP buffer to        form the Mnmalonate complex (complex described in assay        procedure)

Procedure

-   -   1) 2.5 mL of MnP solution was added to spectrophotometer cuvette    -   2) Few drops of each product from laccase experiments (product        from Step 1) were added to cuvette to conduct hydrocarbon        conversion. The concentrations (number of drops) were adjusted        so that the absorption intensities were relatively the same.    -   3) Products from experiments were measured immediately and then        every 15 minutes (up to 1 hour) using the Genesys 10 UV-VIS        spectrophotometer (range 190 nm-1100 nm), purchased from Fisher        Scientific

Chemical Approach: Chelators

A chemical approach using chelators was also used in this example.Chelators act to remove complex ions from the coal structure tosolubilize coal. Three different chelators were used, however, ammoniumtartrate appeared to be the most effective chelator. For theexperiments, ammonium tartrate was used in the solubilization step ofthe process and MnP was then used in the hydrocarbon conversion step ofthe process. A more detailed procedure for this approach is shown below.

Chelator Procedure

Step 1: Ammonium Tartrate Solution

-   -   Chelator Solution—1.00 g of ammonium tartrate was mixed with 250        ml of H2O

Procedure

-   -   1) Coal—0.10 g of each pretreated dried coal sample was used for        each experiment    -   2) 0.1 g of coal combined with 10 ml of chelator solution in        each test tube    -   3) Each test tube was shaken on a Shaker Lab Line Orbital Shaker        overnight    -   4) Products from experiments were measured using the Genesys 10        UV-VIS spectrophotometer (range 190 nm-1100 nm), purchased from        Fisher Scientific. The product concentrations (number of drops)        measured in the spectrophotometer were adjusted so that the        absorption intensities were relatively the same.

Step 2: Manganese Peroxidase

Solutions

-   -   MnP Buffer—160 mM malonic acid solution, pH 4.5    -   MnP solution—MnP oxidizer solution was mixed in MnP buffer to        form the Mnmalonate complex (complex described in assay        procedure)

Procedure

-   -   1) 2.5 mL of MnP solution was added to spectrophotometer cuvette    -   2) Few drops of each product from tartrate experiments (product        from Step 1) were added to cuvette to conduct hydrocarbon        conversion. The concentrations (number of drops) were adjusted        so that the absorption intensities were relatively the same.    -   3) Products from experiments were measured immediately and then        every 15 minutes (up to 1 hour) using the Genesys 10 UV-VIS        spectrophotometer (range 190 nm-1100 nm), purchased from Fisher        Scientific

Enzymatic Approach: Phanerochaete Chrysosporium

In addition to using extracellular enzymes, experiments were conductedusing live organisms known to produce lignin degrading enzymes. Theseexperiments were designed to provide an environment for the liveorganism to excrete such extracellular enzymes to degrade pretreatedcoal. The organism used, Phanerochaete chrysosporium (P. chrysosporium),is a white rot fungi which excretes extracellular enzymes to degradelignin. This organism was purchased from ATCC (ATCC #24725). Twodifferent growth media were used to grow the P. chrysosporium: PotatoDextrose (PD) and Sabouraud Dextrose (SD). A more detailed experimentalprocedure for this approach is shown below.

P. Chrysosporium Procedure

-   -   1) 0.2 g of dried, pretreated coal was weighed and placed into a        50 ml Erlenmeyer flask    -   2) 30 ml of growth media broth (inoculated with P.        chrysosporium) was added to the coal in the flasks    -   3) Each flask was shaken on a Shaker Lab Line Orbital Shaker        constantly for 14 days    -   4) Samples were taken from each flask after the first 2 hours        for an initial reading, as well as once a day on Days 1-7, Day        10, and Day 14. These samples were measured using the Genesys 10        UV-VIS spectrophotometer (range 190 nm-1100 nm), purchased from        Fisher Scientific.

Results. As previously described, three different approaches were takenwhen conducting the solubilization and conversion experiments: (1)laccase and MnP, (2) ammonium tartrate and MnP, and (3) live P.Chrysosporium using two different growth media. The first two approachesincluded a solubilization step, using both enzymes and chelators, toinitially decompose the coal structure; the decomposed coal is passed toa hydrocarbon conversion step, using additional enzymes, to furthercrack the hydrocarbon molecules into smaller hydrocarbon molecules. Inthe live organism approach, P. Chrysosporium was used to excrete anumber of extracellular enzymes to both decompose and then further crackthe coal structure.

After experimentation with enzymes and chelators in the second and thirdquarters, it was determined that the enzymatic approach was moreeffective in degrading bituminous coal than the chemical (chelator)approach. In addition, chelators are expensive and pose potentiallyadverse, environmental impacts. From these experimental results andenvironmental considerations, it was determined that the chelatorapproach was not optimal.

The experiments with live P. Chrysosporium were conducted usingdifferent types of growth media. Results from these experiments showedthat the optimal growth conditions were the PD growth medium and the SDgrowth medium, both with the least amount of peptone added.

Successful results of coal degradation were obtained from both theenzymatic approach (laccase and MnP) and live organism approach (P.Chrysosporium). Therefore, only the spectral results from theseapproaches are presented and analyzed. FIGS. 19A through 22C show theresults from these experiments.

Each curve shows wavelength (nm) vs. relative absorbance (A), whererelative absorbance (A) is related to transmittance (T): A=log lO (1/T).The legends on the graphs should be read left to right, showing theorder in which samples were measured.

The results are divided into sections according to the type ofpretreatment used. Only the spectral results from the most effectivepretreatments are presented. These pretreatments are hydrogen peroxide(3%, 15%, and 30%), heated coal, mine water, and nitric acid (pH 1)pretreatments. After a comparison of these results the best overallapproach for degradation of bituminous coal will be determined.

3% H2O2 Pretreated Coal

FIG. 19A illustrates the results from the 3% H2O2, pretreated coalexperiments using the laccase and MnP solutions. The spectrum for thepretreated coal in laccase buffer shows a peak around 215 nm and anabsorbance of about 1.0 A (red curve). This spectrum can be compared tothe spectrum of the pretreated coal in laccase solution, which shows apeak around 215 nm and an absorbance of about 2.0 A (blue curve).

These two spectra show that the 3% H2O2 pretreatment does have someeffect on solubilizing the coal, but the laccase works to furtheroxidize and solubilize it. The spectra in FIG. 19A also illustrate ashift in peaks from 215 nm to about 244 nm and a decrease in absorbancefrom 2.0 A to 1.74 A, which occurs when the laccase product was added tothe MnP solution (green curve—0 min). The absorbance (green curve—0 min)continues to decrease over time to about 1.45 A (pink curve—15 min,turquoise curve—30 min, yellow curve—45 min, gray curve—1 hr).

FIG. 19B displays the results from the 3% H2O2, pretreated coalexperiments. The spectrum for the untreated coal in tartrate solutiondoes not exhibit any strong absorbance peaks in the spectra (red curve).However, the pretreated coal in tartrate solution shows a peak around225 nm and an absorbance of about 0.634 A (blue curve). This shows thepretreatment has a very strong effect on the chemical solubilization ofthe coal using tartrate solution. The spectra in FIG. 19B alsoillustrate a shift in peaks from 225 nm to about 243 nm, which occurswhen the tartrate product was added to the MnP solution (green curve—0min). This absorbance (green curve—0 min) continues to decrease overtime to about 0.286 A (pink curve—15 min, turquoise curve—30 min, yellowcurve—45 min, gray curve—1 hr).

FIG. 19C illustrates the results from the 3% H2O2, pretreated coalexperiment in PD growth medium containing P. chrysosporium. The spectrumfor the pretreated coal in these growth conditions initially displays apeak around 245 nm with an absorbance of about 1.30 A and a peak around290 nm with an absorbance of about 2.10 A (red curve). These peaks donot shift in wavelength (nm) over time, but the absorbance (A) each daydoes tend to change slightly. On Day 1, the absorbance decreases toabout 0.52 A at 245 nm and 1.60 A at 290 nm (blue curve), but increasesto about 1.45 A at 245 nm and 2.20 A at 290 nm on Day 2 (green curve).After Day 2, the absorbance decreases again on Day 3 to 1.28 A at 245 nmand 1.35 A at 290 nm (pink curve). Absorbance then continues to increasefor the remaining days. Day 4: 1.36 A at 245 nm and 1.58 A at 290 nm(turquoise curve); Day 5: 1.51 A at 245 nm and 2.02 A at 290 nm (yellowcurve); Day 6: 1.56 A at 245 nm and 2.48 nm at 290 nm (gray curve); Day7: 1.52 A at 245 nm and 2.63 A at 290 nm (black curve).

FIG. 19D shows the results from the 3% H2O2, pretreated coal experimentin SD growth medium, with one tenth the amount of peptone (0.1×),containing P. chrysosporium. The spectrum for the pretreated coal inthese growth conditions initially displays a peak around 245 nm and anabsorbance of 2.73 A (red curve). This peak at 245 nm does not shift inwavelength and does not increase or decrease significantly in absorbancefrom Day 1 through Day 10. Day 1: 2.52 A (blue curve); Day 2: 2.57 A(green curve); Day 3: 2.42 A (pink curve), Day 4: 2.39 A (turquoisecurve); Day 5: 2.71 A (yellow curve); Day 6: 2.66 A (gray curve); Day 7:2.52 A (black curve); Day 10: 2.60 A (orange curve). The absorbance at245 nm then decreases on Day 14 to 2.02 A (beige curve).

15% H2O2 Pretreated Coal

FIG. 19E shows the results from the 15% H2O2, pretreated coalexperiments. The spectrum for the pretreated coal in laccase buffershows a peak around 230 nm and an absorbance of only about 0.877 A (redcurve). This spectrum can be compared to the spectrum of the pretreatedcoal in laccase solution, which shows a peak around 224 nm and anabsorbance of about 3.20 A (blue curve). By comparing these spectra, itcan be seen that the pretreatment does have an impact on thesolubilization of the coal, but that the laccase is needed for furthersolubilization. The spectra in FIG. 19E also illustrate a shift in peaksfrom 224 nm to about 249 nm and a decrease in absorbance from 3.20 A to1.99 A, which occurs when the laccase product was initially added to theMnP solution (green curve—0 min). This absorbance (green curve—0 min)continues to decrease over time to about 1.63 A (pink curve—15 min,turquoise curve—30 min, yellow curve—45 min, gray curve—1 hr).

FIG. 19F shows the results from 15% H2O2, pretreated coal experiments.The spectrum for the untreated coal in tartrate solution does notexhibit any strong absorbance peaks in the spectra (red curve). However,the pretreated coal in tartrate solution shows a peak around 227 nm andan absorbance of about 1.21 A (blue curve). This indicates that thepretreatment has a very strong impact on the chemical solubilization ofthe coal in tartrate solution. The spectra in FIG. 19F also illustrate ashift in peaks from 227 nm to about 245 nm and a slight drop inabsorbance from 1.21 A to 1.14 A, which occurs when the tartrate productwas initially added to the MnP solution (green curve—0 min). Thisabsorbance (green curve—0 min) continues to decrease over time to about0.52 A (pink curve—15 min, turquoise curve—30 min, yellow curve—45 min,gray curve—1 hr).

FIG. 19G shows the results from the 15% H2O2, pretreated coal experimentin PD growth medium containing P. chrysosporium. The spectrum (redcurve) for the pretreated coal in these conditions initially shows twopeaks: one peak at a wavelength of 245 nm with absorbance of 1.00 A andone peak at a wavelength of 290 nm with absorbance of 2.20 A. The peaksin FIG. 19G do not appear to shift in wavelength but do show changes inthe absorbance from day to day. After the first day, the peak absorbancedecreases from the initial peak to about 0.69 A at 245 nm and 1.80 A at290 nm (blue curve). This absorbance then increases on Day 2 to 1.56 Aat 245 nm and 2.30 A at 290 nm (green curve) and decreases on Day 3 to1.31 A at 245 nm and 1.35 A at 290 nm (pink curve). After Day 3, thecurves then increase in absorbance for the remaining days. Day 4: 1.38 Aat 245 nm and 1.58 A at 290 nm (turquoise curve); Day 5: 1.53 A at 245nm and 2.13 A at 290 nm (yellow curve); Day 6: 1.62 A at 245 nm and 2.22A at 290 nm (gray curve); Day 7: 1.74 A at 245 nm and 2.69 A at 290 nm(black curve); Day 10: 1.75 A at 245 nm and 2.76 A at 290 nm (orangecurve).

FIG. 19H shows the results from the 15% H2O2, pretreated coal experimentin SD growth medium, with one tenth the amount of peptone (0.1×),containing P. chrysosporium. The spectrum for the pretreated coal inthese growth conditions initially displays a peak around 245 nm and anabsorbance of 2.78 A (red curve). This peak at 245 nm does not shift inwavelength but decreases slightly in absorbance from Day 1 through Day14. Day 1: 2.64 A (blue curve); Day 2: 2.45 A (green curve); Day 3: 2.38A (pink curve), Day 4: 2.39 A (turquoise curve); Day 5: 2.02 A (yellowcurve); Day 6: 2.09 A (gray curve); Day 7: 2.12 A (black curve); Day 10:2.13 A (orange curve); Day 14: 1.50 A (beige curve).

30% H2O2 Pretreated Coal

FIG. 19I shows the results from the 30% H2O2, pretreated coalexperiments. The spectrum for the pretreated coal in laccase buffershows a peak around 224 nm and an absorbance of about 1.99 A (redcurve). This spectrum can be compared to the spectrum of the pretreatedcoal in laccase solution, which shows a peak around 211 nm and anabsorbance of about 2.58 A (blue curve). The comparison of these spectraillustrates that the 30% pretreatment by itself is almost as effectiveas the laccase in solubilizing the coal. The spectra in FIG. 19I alsoillustrate a shift in peaks from 211 nm to about 249 nm and a decreasein absorbance from 2.58 A to 1.77 A, which occurs when the laccaseproduct was initially added to the MnP solution (green curve). Thisabsorbance (green curve) also continues to decrease over time to about1.13 A (pink curve—15 min, turquoise curve—30 min, yellow curve—45 min,gray curve—1 hr).

FIG. 19J shows the results from the 30% H2O2, pretreated coalexperiments. The spectrum for the untreated coal in tartrate solutiondoes not exhibit any strong absorbance peaks in the spectra (red curve).However, the pretreated coal in tartrate solution shows a peak around228 nm and an absorbance of about 1.52 A (blue curve). The difference inthese peaks shows the pretreatment is necessary to chemically solubilizethe coal in tartrate solution. The spectra in FIG. 19J also illustratesa shift in peaks from 228 nm to about 245 nm and a drop in absorbancefrom 1.52 A to 1.18 A, which occurs when the tartrate product was addedto the MnP solution (green curve—0 min). This absorbance (green curve—0min) continues to decrease over time to about 0.87 A (pink curve—15 min,turquoise curve—30 min, yellow curve—45 min, gray curve—1 hr).

FIG. 19K shows the results from the 30% H2O2, pretreated coal experimentin PD growth medium containing P. chrysosporium. The spectrum for thisparticular pretreated coal initially shows a peak around 245 nm with anabsorbance of about 0.86 A and a peak around 295 nm with an absorbanceof 1.89 A (red curve). After the first day, the absorbance drops toabout 0.59 A at 245 nm, but remains at about 0.86 A at 295 nm (bluecurve). From Day 2 to Day 5, the absorbance at 245 nm does not changesignificantly, while the peak at 295 nm changes slightly each day. Day2: 1.54 A at 245 nm and 2.56 A at 295 nm (green curve); Day 3: 1.38 A at245 nm and 2.22 A at 295 nm (pink curve); Day 4: 1.57 A at 245 nm and2.26 A at 295 nm (turquoise curve); Day 5: 1.76 A at 245 nm and 2.83 Aat 295 nm (yellow curve). On Day 6, the absorbance increases at bothpeaks to about 2.29 A at 245 nm and 3.46 A at 295 nm (gray curve).

FIG. 19L shows the results from the 30% H2O2, pretreated coal experimentin SD growth medium, with one tenth the amount of peptone (0.1×),containing P. chrysosporium. The spectrum for the pretreated coal inthese growth conditions initially displays a peak around 243 nm and anabsorbance of 1.56 A (red curve). This peak at 245 nm does not shift inwavelength but decreases in absorbance over Day 1 through Day 7, withthe exception of Days 3 and 10 which increase in absorbance. Day 1: 1.47A (blue curve); Day 2: 1.39 A (green curve); Day 3: 2.09 A (pink curve),Day 4: 1.30 A (turquoise curve); Day 5: 1.07 A (yellow curve); Day 6:0.93 A (gray curve); Day 7: 0.93 A (black curve). On Day 10, theabsorbance at 245 nm increases to 2.58 A (orange curve).

Heated Coal Pretreatment

FIG. 19M shows the results from the heated, 30% H2O2, pretreated coalexperiments. The spectrum for the pretreated coal in laccase buffershows a peak around 237 nm and an absorbance of only about 1.41 A (redcurve). This spectrum can be compared to the spectrum of the pretreatedcoal in laccase solution, which shows a peak around 227 nm and anabsorbance of about 3.09 A (blue curve). The comparison of these spectrashows that the pretreatment does have some effect on the coalsolubilization, but that the laccase further oxidizes the coal forsolubilization. The spectra in FIG. 19M also illustrate a shift in peaksfrom 227 nm to about 247 nm and a decrease in absorbance from 3.09 A to2.31 A, which occurs when the laccase product was initially added to theMnP solution (green curve—0 min). This curve also continues to changeover time (pink curve—15 min, turquoise curve—30 min, yellow curve—45min, gray curve—1 hr).

FIG. 20A shows the results from the heated coal experiment. The spectrumfor this pretreatment in PD growth medium with P. chrysosporium does notinitially display a strong peak on Day 0 (red curve) or Day 1 (bluecurve). A peak then starts to form on Day 2 around 240 nm with anabsorbance of 1.39 A (green curve). This peak at 240 nm then decreasesin absorbance on Day 3 to 0.68 A (pink curve) and Day 4 to 0.23 A(turquoise curve). Finally, the peak increases in absorbance at 240 nmover the last 2 days. Day 5: 1.39 A at 245 nm (yellow curve) and Day 6:1.93 A at 240 nm (gray curve).

FIG. 20B shows the results from the heated coal experiment in SD growthmedium, with one tenth the amount of peptone (0.1×), containing P.chrysosporium. The spectrum for the pretreated coal in these conditionsinitially shows a peak at about 240 nm and an absorbance of 0.61 A (redcurve). This peak at 240 nm then increases in absorbance to about 0.78 Aon Day 1 (blue curve). From Day 2 through Day 5, the absorbance at thiswavelength continues to change. Day 2: 0.73 A (green curve); Day 3: 0.56A (pink curve); Day 4: 0.78 A (turquoise curve); Day 5: 0.44 A (yellowcurve). On Day 6 the wavelength appears to shift to about 260 nm andremains at this wavelength on Day 7 and Day 10. Day 6: 0.66 A (graycurve); Day 7: 0.55 A (black curve) and Day 10: 0.95 A (orange curve).

Mine Water Pretreated Coal

FIG. 21A illustrates the results from the mine water, pretreated coalexperiments. The spectrum for the pretreated coal in laccase buffershows a peak around 245 nm and an absorbance of about 0.71 A (redcurve). This spectrum can be compared to the spectrum of the pretreatedcoal in laccase solution, which shows a peak around 217 nm and anabsorbance of about 2.34 A (blue curve). The comparison of these spectrashows that this pretreatment does help to initially oxidize anddecompose the coal, but that the laccase is necessary for furtherdecomposition. The spectra in FIG. 21A also illustrate a shift in peaksfrom 217 nm to about 249 nm and a slight decrease in absorbance from2.58 A to 2.25 A, which occurs when the laccase product was initiallyadded to the MnP solution (green curve—0 min). This absorbance (greencurve—0 min) continues to decrease over time to about 1.83 A (pinkcurve—15 min, turquoise curve—30 min, yellow curve—45 min, gray curve—1hr).

FIG. 21B shows the results from the mine water, pretreated coalexperiments. The spectrum for the untreated coal in tartrate solutiondoes not exhibit any strong absorbance peaks in the spectra (red curve).However, the pretreated coal in tartrate solution shows a peak around228 nm and an absorbance of about 1.43 A (blue curve). The comparison ofthese two curves shows that the pretreatment is necessary to chemicallysolubilize the coal in tartrate solution. The spectra in FIG. 21B alsoillustrate a shift in peaks from 228 nm to about 245 nm, which occurswhen the tartrate product was added to the MnP solution (green curve—0min). This absorbance (green curve—0 min) continues to decrease overtime to about 0.87 A (pink curve—15 min, turquoise curve—30 min, yellowcurve—45 min, gray curve—1 hr).

FIG. 21C illustrates the results from the mine water, pretreated coalexperiment in PD growth medium containing P. chrysosporium. The spectrumfor this pretreated coal shows 2 peaks: one peak around 245 nm and onepeak around 305 nm. The peak at 245 nm initially has an absorbance of1.17 A (red curve) and remains at 1.17 A after Day 1 (blue curve). Thepeak at 245 nm then increases in absorbance on Day 2 to about 2.00 A(green curve) and only changes slightly in absorbance over the remainingdays. Day 4: 1.84 A (pink curve); Day 5: 1.89 A (turquoise curve); Day6: 1.87 A (yellow curve); Day 7: 1.78 A (gray curve); Day 10: 1.82 A(black curve). The peak at 305 nm has an initial absorbance of 2.97 A(red curve) and also changes very slightly over the time period. Day 1:3.06 A (blue curve); Day 2: 3.51 A (green curve); Day 4: 3.49 A (pinkcurve); Day 5: 3.12 A (turquoise curve); Day 6: 3.31 A (yellow curve);Day 7: 3.24 A (gray curve); Day 10: 3.06 A (black curve).

FIG. 21D shows the results from the mine water, pretreated coalexperiment in SD growth medium, with one tenth the amount of peptone(0.1×), containing P. chrysosporium. The spectrum for the pretreatedcoal in these conditions initially shows a broad peak around 297 nm withan absorbance of about 3.94 A (red curve). The spectra for Day 1 throughDay 5 remains relatively unchanged at 297 nm. Day 1: 3.95 A (bluecurve); Day 2: 3.96 A (green curve); Day 3: 3.89 A (pink curve); Day 4:3.97 A (turquoise curve); Day 5: 3.82 A (yellow curve). On Day 6 theabsorbance begins to decrease slightly at 297 nm and continues todecrease slightly on Day 7 and Day 10. Day 6: 3.70 A (gray curve); Day7: 3.60 A (black curve); Day 10: 3.45 A (orange curve). On Day 14, theabsorbance increases to 3.76 A (beige curve).

HNO3 (pH 1 Pretreated Coal

FIG. 22A shows the results from the HNO3 (pH 1), pretreated coalexperiments. The spectrum for the pretreated coal in laccase buffershows a peak around 210 nm and an absorbance of about only 0.47 A (redcurve), and can be compared to the spectrum for the pretreated coal inlaccase solution, which shows a peak around 213 nm and an absorbance ofabout 1.68 A (blue curve). The comparison of these curves indicates thatthe pretreatment does have an effect on coal solubilization, but thatthe laccase solution works to further oxidize and decompose the coal.The spectra in FIG. 22A also illustrate a shift in peaks from 213 nm toabout 244 nm and a decrease in absorbance from 1.68 A to 1.35 A, whichoccurs when the laccase product was initially added to the MnP solution(green curve—0 min). This absorbance (green curve—0 min) continues todecrease over time to about 0.99 A (pink curve—15 min, turquoisecurve—30 min, yellow curve—45 min, gray curve—1 hr).

FIG. 22B shows the results from the HNO3 (pH 1), pretreated coalexperiment in PD growth medium containing P. chrysosporium. The spectrumfor this experiment initially shows a peak around 260 nm with a relativeabsorbance of −0.52 A (red curve). This absorbance at 260 nm increasesin absorbance to −0.32 A on Day 1 (blue curve). On Day 2 the peak shiftsand begins to form at 240 nm with an absorbance of 0.794 A (greencurve). This peak at 240 nm slightly increases on Day 3 to 0.93 A (pinkcurve), but then continues to decrease in absorbance from Day 4 to Day7. Day 4: 0.80 A (turquoise curve); Day 5: 0.49 A (yellow curve); Day 6:0.44 A (gray curve); Day 7: 0.003 A (black curve).

FIG. 22C shows the results from the HNO3 (pH 1), pretreated coalexperiment in SD growth medium, with one tenth the amount of peptone(0.1×), containing P. chrysosporium. The spectrum for the pretreatedcoal in these conditions initially shows an absorbance of −0.23 A around235 nm (red curve). The absorbance at this wavelength then continues todecrease on Day 1 through Day 7. Day 1: −0.35 A (blue curve); Day 2:−0.44 A (green curve); Day 3: −0.34 A (pink curve); Day 4: −0.33 A(turquoise curve); Day 5: −0.55 A (yellow curve); Day 6: −0.70 A (graycurve); Day 7: −0.78 A (black curve). Finally on Day 10, the absorbanceat 235 nm increases to about −0.30 A (orange curve).

The work for this example focused on conversion of bituminous coal toliquid fuels. This process involves the same steps taken by naturalmicroorganisms to break down the molecular structures found in coal intouseable forms that can enter their metabolic pathways for the release ofcarbon and energy. One example of such a pathway is the digestion ofphenol, given in FIG. 23.

Phenols represent an important class of functional groups since 40-75%of the carbon in bituminous coal is aromatic, and phenols and phenolicmoieties represent the preferred entry point for microbial aromatic ringcleavage.

Opening aromatic ring structures is also a necessary first step in theconversion of coal to liquid fuels in the CFS process. For reference, atime series of UV spectra for phenol degradation is given in FIG. 24.While these results were not obtained from a biological system, they doprovide a qualitative measure of the transitions in UV spectra that canbe expected from the reaction pathway shown above.

As the graph shows, absorbance at the front end of the UV spectraincreases during the initial stages of degradation corresponding to theformation of aromatic intermediates. As more intermediates are formed,the concentration of phenol decreases, shown by the decrease inabsorbance for phenol at 268 nm. Both peaks then begin to decrease asthe phenol is consumed and the aromatic intermediates undergo ringcleavage. Eventually absorbance approaches zero as the cleaved aromaticintermediates are converted to organic acids.

The spectra presented in this example show that the methods used in thisexample produce the same type of degradation as shown in the UV spectrafor phenol degradation. It is also clear that the extent to which coalundergoes conversion varies significantly with the way the coal ispretreated.

As previously described, three different approaches were taken todegrade bituminous coal: (1) laccase and MnP, (2) tartrate and MnP, and(3) live P. chrysosporium using two different growth media. The resultsfrom the laccase and MnP experiments and live P. chrysosporiumexperiments show the best results and are compared.

These spectra are shown in FIGS. 19A through 22C. As seen in FIGS. 19Athrough 22C, the spectra for these approaches using variouspretreatments of coal display peaks in the 240 nm to 300 nm region.These peaks correspond to the smaller aromatic ring intermediates andorganic acids formed from the degradation of bituminous coal. From thesespectra, it is apparent that both of the methods do work to degradecoal.

The first enzymatic solubilization and conversion experiments wereconducted using laccase and MnP, shown in FIGS. 19A, 19E, 19I, 19M, 21A,and 22A. The spectra show peaks around 245 nm-250 nm. The peaks in thesespectra start to decrease in absorbance over a period of 1 hour,corresponding to the consumption of aromatic intermediates as theyundergo ring cleavage.

Live P. chrysosporium was used as another method to degrade bituminouscoal. It was grown in two different growth media to provide sufficientgrowth conditions for the organism to excrete extracellular enzymes todegrade coal. The spectra from the potato dextrose (PD) growth mediumcontaining P. chrysosporium display peaks in the 240 nm to 300 nmregion, shown in FIGS. 19C, 19G, 19K, 20A, 21C, and 22B. The peakabsorbance is shown to decrease over a period of 7 to 10 days in theseexperiments.

P. chrysosporium was also grown in a Sabouraud Dextrose (SD) growthmedium. The spectra corresponding to these experiments are shown inFIGS. 19D, 19H, 19L, 20B, 21D, and 22C. Most of the peaks in the spectraare seen around 235 nm to 245 nm. A decrease in peak absorbance can beseen over a period of 10 to 14 days.

The main parameters compared between the UV-VIS spectra for the twodifferent enzymatic methods were the location of the peaks (nm) and thedecrease in peak absorbance (A). As mentioned, the peaks from theexperiments fall in the range of 240 nm to 300 nm, which correspond toaromatic intermediates formed when breaking down the coal structure. Thepeaks from the laccase+MnP and P. chrysosporium in the S.D. growthmedium experiments show similar peaks in the 235 nm to 250 nm region.The peaks from the P. chrysosporium in the P.D. growth medium show twodifferent peaks, in the 245 nm region and in the 290 nm region.

When comparing the decreases in peak absorbance, it can be seen that theexperiments conducted with laccase and MnP experiments decrease inabsorbance over the time period of 1 hour. However, in the live P.chrysosporium experiments, the decrease in peak absorbance is seen overa period of 10 to 14 days. This is a significant difference in the timeit takes for the aromatic intermediates to form and ring cleavage tooccur, and illustrates one of the major differences between usingextracellular enzymes and using live organisms.

When comparing the spectra among the different types of pretreatment, itcan be seen that the most consistent results are seen in the 3%, 15%,and 30% hydrogen peroxide pretreatments. These spectra show peaks in the235 nm to 240 nm region for all of the pretreated coal samples. In thespectra for the heated coal, the peaks for the experiments using live P.chrysosporium are not as consistent as the peaks for the experimentsusing extracellular enzymes. In the mine water pretreated coal, thepeaks do not decrease in absorbance over time in the experiments usinglive P. chrysosporium as they do in the experiments using extracellularenzymes. Finally, the spectra for the nitric acid pretreated coal shownegative absorbance in the experiments using live P. chrysosporium,which is not consistent with the peaks obtained using extracellularenzymes.

In this example, it has been shown that both extracellular enzymes andlive organisms can be used to decompose bituminous coal. The spectrafrom these experiments show peaks in 240 nm to 300 nm region,corresponding to the smaller aromatic ring intermediates and organicacids formed from the degradation coal. However, when comparing thesedifferent methods, it is apparent that the most efficient way to attackthe coal structure is with enzymes. This process has a few advantagesover using live organisms.

One of the main advantages of using extracellular enzymes instead oflive organisms is the time necessary for decomposition. As seen in thespectra, the results from the experiments using the laccase andmanganese peroxidase showed a decrease in absorbance within the firsthour of experimentation. Similar decreases in absorbance are also seenin the P. chrysosporium results. However, these results were recordedover days, rather than minutes. It obviously takes a longer time for thelive organism to excrete the proper extracellular enzymes to degradecoal.

In addition, as mentioned before, working with organisms is difficultbecause they are alive and the number of unknowns is numerous. Thisincludes maintaining the ideal culture conditions for the organism andalso controlling contaminants and fungicidal byproducts that may preventthe organism from growing or excreting extracellular enzymes to breakdown coal.

Other ligninolytic enzymes, such as lignin peroxidase, may also be usedto degrade bituminous coal. In addition, other types of white rot fungi,such as Trametes Versicolor, may be grown and exposed to bituminouscoal. Different culture conditions may also affect how the organismsexcrete extracellular enzymes to degrade coal.

While the preferred embodiments of the invention have been shown anddescribed, modifications thereof can be made by one skilled in the artwithout departing from the spirit and teachings of the invention. Theembodiments described and the examples provided herein are exemplaryonly, and are not intended to be limiting. Many variations andmodifications of the invention disclosed herein are possible and arewithin the scope of the invention. Accordingly, the scope of protectionis not limited by the description set out above, but is only limited bythe claims that follow, the scope including all equivalents of thesubject matter of the claims.

We claim:
 1. A method for producing liquid hydrocarbon productcomprising, disintegrating a hydrocarbon source; pretreating thedisintegrated hydrocarbon source; solubilizing the disintegratedhydrocarbon source to form a slurry comprising a reactant molecule ofthe hydrocarbon source; admixing a biochemical liquor into the slurry,wherein the biochemical liquor comprises at least one conversion enzymeconfigured to facilitate bond selective photo-fragmentation of saidreactant molecule of the hydrocarbon source, to form liquid hydrocarbonsvia enzyme assisted bond selective photo-fragmentation, wherein saidconversion enzyme comprises reactive sites configured to restrict saidreactant molecule such that photo-fragmentation favorably targets apreselected internal bond of said reactant molecule; separating theliquid hydrocarbons from the slurry, wherein contaminants remain in theslurry; and enriching the liquid hydrocarbons to form a liquidhydrocarbon product.
 2. The method of claim 1, wherein disintegratingthe hydrocarbon source comprises comminution of the hydrocarbon source.3. The method of claim 2, wherein comminution comprises grinding.
 4. Themethod of claim 2, wherein comminution comprises high-pressure steamtreatment.
 5. The method of claim 1, wherein pretreating thedisintegrated hydrocarbon source comprises chemical pretreatment, heatpretreatment, oxidation of the hydrocarbon source, or a combinationthereof.
 6. The method of claim 1, wherein solubilizing thedisintegrated hydrocarbon source comprises treating the disintegratedhydrocarbon source with at least one enzyme.
 7. The method of claim 1,wherein admixing biochemical liquor comprises admixing at least oneadditional enzyme.
 8. The method of claim 7, wherein admixing at leastone additional enzyme further comprises admixing an enzyme forconverting a hydrocarbon source to lower molecular weight hydrocarbons.9. The method of claim 1, wherein separating liquid hydrocarbonscomprises a process of settling the slurry from the liquid hydrocarbon.10. The method of claim 1, wherein separating liquid hydrocarbonscomprises settling contaminants from the liquid hydrocarbon.
 11. Themethod of claim 1, wherein enriching the liquid hydrocarbon comprisesadmixing the liquid hydrocarbon with at least one enzyme.
 12. The methodof claim 1, wherein the biochemical liquor comprises a modified enzyme.13. The method of claim 12, wherein the modified enzyme comprises anenzyme that is genetically modified.
 14. The method of claim 12, whereinthe modified enzyme comprises an enzyme that is chemically modified. 17.The method of claim 1, wherein the method is conducted in-situ in a coalmine or ex-situ on mined coal.
 18. The method of claim 1, whereinenriching the liquid hydrocarbons comprises improving the liquidhydrocarbon product qualities prior to distillation.
 19. The method ofclaim 1, wherein the liquid hydrocarbon product comprises at least oneselected from the group consisting of gasoline, diesel, kerosene, anddistillates thereof.
 20. The method of claim 1, wherein the hydrocarbonsource comprises at least one selected from the group consisting ofcoal, anthracite coal, bituminous coal, lignite, sub-bituminous coal,low-rank coals, coke, tar sand, and oil shale.